Factor VII is a vitamin K dependent protein which associates with Tissue Factor and initiates the extrinsic pathway of coagulation. Elevated plasma levels of Factor VII may be an independent risk factor for ischemic heart disease, and have been associated with other hypercoagulable states. This proposal is designed to identify and characterize the transcriptional regulatory and termination regions of the factor VII gene, followed by the purification and characterization of novel transcription factors. The experimental approach can be outlined in the following steps: I. DNA STUDIES A. Characterization of the factor VII promoter. l. Functional reporter gene assays of the promoter region. 2. Deletion mutagenesis of the promoter region. 3. Orientation and positional effects of the promoter. 4. DNase I footprinting and methylation interference studies. 5. Site-directed mutagenesis of promoter sequences. B. Search for additional regulatory elements. l. Extend 5'-flanking untranslated sequence by chromosome walking. 2. Reporter gene constructs with the first intron of the factor VII gene. II. DETERMINATION OF THE TRANSCRIPTION TERMINATION REGION: By northern analysis of cellular mRNA transcripts derived from reporter gene constructs. III. PURIFICATION AND CHARACTERIZATION OF TRANSCRIPTION FACTORS A. Development of gel-shift assay to identify optimal conditions for DNA-binding. B. UV cross-linking studies to identify the factor(s) which interact with the promoter sequences. C. DNA-affinity chromatography. D. cDNA cloning and expression of the transcriptional factor(s) The characterization of the transcriptional regulatory region and the isolation of their sequence-specific nuclear factors will expand our current understanding of tissue-specific gene regulation. This information may also help determine if the vitamin K-dependent coagulation proteins are regulated by a common nuclear factor(s) and may give insight into the molecular events which trigger the elevation of plasma levels of factor VII in hypercoagulable states such as ischemic heart disease.